Mycoplasma pulmonis infections in laboratory rodent colonies is a serious problem in medical research. Its pathogenic mechanisms are unknown, but previous studies in this laboratory indicated that virulence may be related to the activities of specific membrane proteins, specifically membrane bound hemolysis (cytolysin) and a membrane bound endonuclease. During the last grant period, the membrane bound hemolysin was identified as an 88.5 kDa protein by using monoclonal antibodies (Mabs) in radioimmune precipitation assays. In addition, techniques were developed to construct defined mutations in specific locations (i.e. within specific genes) in the mycoplasmal chromosome. These techniques will allow the analysis of the contribution of specific gene products to virulence. Previous approaches to mycoplasmal virulence and used spontaneous or chemically- induced mutant. The proposed studies will use the transposons, Tn916 and Tn4001, as well as integrative plasmid vectors derived from their antibiotic resistances, to introduce single mutations in putative virulence associated activities in M. pulmonis. To further study the contribution of the membrane hemolysin and nuclease to virulence we propose first to identify the membrane nuclease by using immunological approaches (i.e. Mabs). Both the hemolysin and the nuclease will be purified using affinity chromatography and monospecific polyclonal antisera produced against each of the proteins. The gene sequences for both proteins will be identified by using he polyclonal antisera and Mabs to screen genomic libraries. Restriction enzyme maps will be constructed of each gene and interposon mutations made to each activity by using the integrative plasmids and transposons. These mutants will be examined for location of he insert and loss of activity. In vitro and in vivo assays will be used to determine the role these proteins play in cytotoxicity (hemolysin) uptake and utilization of nucleic acid precursors (nuclease) and virulence (both). Additional membrane proteins identified in these studies will be examined for their potential role in pathogenesis.